Normal cellular differentiation and development rely extensively on silencing mechanisms. Disruption of silencing can lead to genetic disorders, chromosome loss and contribute to tumorigenesis. Our long term goal is to determine the normal mechanisms that lead to establishment and maintenance of silenced regions (heterochromatin) of the genome. The goal of this proposal is to define how heterochromatin at centromeres is established. A major impediment to research on the establishment of heterochromatin has been difficulty in separating establishment from maintenance. To circumvent this problem, we utilize the exquisite genetic tractability of the fission yeast, where silenced chromatin shares many features with mammals, including reliance on similar chromatin marking systems and a shared dependence on non-coding RNA and the RNAi pathway for silencing control. Through characterization of a macromolecular complex that is important for the assembly of heterochromatin, we generated a novel mutant strain of yeast that can maintain pre-assembled heterochromatin, but cannot support its de-novo establishment. We will use this mutant to screen genes that are known to play a role in heterochromatin stability to identify those that are required for heterochromatin establishment. We have already found that one establishment gene that encodes a histone methyltransferase, Clr4, (the homolog of Suv39 proteins in flies and mammals) which methylates histone H3 on lysine 9 (K9) only in regions of heterochromatin. We anticipate that other establishment genes will regulate Clr4's recruitment to chromatin and / or its activity. Second, we will determine the DNA sequences that are required for the establishment of heterochromatin. We will define hot-spots for Clr4 activity using high resolution chromatin immunoprecipitation analyses, and will assess whether these sequences suffice to establish de novo heterochromatin. We will determine which establishment genes work through which establishment sequences. Finally, we will determine if K9 methylation of histone H3 suffices to establish heterochromatin, and the role that the proteins that bind this mark play in the establishment of heterochromatin. These studies will yield a basic knowledge of the mechanisms that dictate heterochromatin establishment and will provide a framework for future work directed towards understanding and developing therapies for aberrant heterochromatin assembly and disease-related silencing defects in humans. PUBLIC HEALTH RELEVANCE: The turning-off or silencing of specific regions of chromosomes is as important for the normal growth of cells in an organism as is turning-on genes. Silencing is important for cell-type identity and for enabling all cells to divide normally, such that improper silencing can contribute to genetic disorders and cancer. The proposed studies are directly relevant as they will yield a basic knowledge of silencing and help direct future work towards understanding and developing therapies for disease- related silencing defects in humans.